Screening For Arylacetonitrilase And Its Application To The Synthesis Of P-Methoxyphenylacetic Acid

Biotransformation of nitriles has been playing more and more important roles in affording amides and acids.Thus,nitrile-converting enzymes have evoked substantial attention and they are becoming more and more demanding.The product of this paper,p-methoxyphenylacetic acid（MOPAA）,is an important free chemical and pharmaceutical intermediate of high value,whose synthesis mostly depends on chemical processes at present.Enzymatic hydrolysis of nitriles offers alternatives to conventional nitrile hydrolysis due to the mild conditions and ecofriendly nature,with high yields and meets the demands of "green chemistry".The paper focused on the bioconversion process of p-methoxyphenylacetonitrile（MOPAN）to MOPAA and investigated in terms of the screening,identification,characterization,cultivation and application of the biocatalyst in detail.A high-throughput screening method was established employing p-hydroxyphenylacetonitrile as sole nitrogen source through conventional enrichment culture and then thin layer chromatography（TLC）in the rapid detection of p-hydroxyphenylacetic acid.With this method,11 microorganisms bearing nitrilase activity were selected that were capable of converting MOPAN to MOPAA,among which,strain ZJB-063,with the maximum nitrilase activity,was chosen as the best strain for further studies.Based on morphology,physiological tests,Biolog and the 16S rDNA sequence,strain ZJB-063 was identified as B.subtilis.The paper was the first to describe a nitrilase enzyme from Bacillus subtilis.The medium composition was optimized by single factors and response surface methodology（RSM）.The optimized medium composition was as follows（g/l）:glucose 12.6,yeast powder 6.6, （NH₄）₂SO₄ 5.5,K₂HPO₄ 0.5,KH₂PO₄ 0.5,MgSO₄ 0.5,FeSO₄·7H₂O 0.01. The optimum conditions for cell growth and formation of nitrilase were as follows:initial pH value,7.0;30℃;inoculum volume,4%（v/v）; medium volumetric ratio,20%（v/v）.Under these conditions,a specific nitrilase activity of 17.24 U/g_DCWwas achieved after cultivation for 40 h.Influences of reaction conditions on nitrilase activity were aiso evaluated.It was indicated that the nitrilase exhibited maximal activity in phosphate buffer（pH 7.0）at 32℃.Both MOPAN and MOPAA inhibited the hydrolysis at concentrations above 15 mM.The half-life(t_1/2)of intracellular nitrilase at 30(t_1/292.6 h)and 35℃(t_1/224.0 h)was determined,suggesting that the thermostability of the nitrilase in B. subtilis ZJB-063 was low.The addition of metal ions could not enhance the enzyme activity.The thiol binding reagents CuCl₂ and AgNO₃ were strong inhibitors of the nitrilase activity,which resulted in 84.21 and 92.26%inhibition,respectively,suggesting that one or more thiol residues were necessary for this enzyme.The addition of 5%methanol and DMSO in the reaction mixture resulted in approximately 35.15 and 62.70%,respectively,enhancement in nitrilase activity.Under the optimized conversion conditions,10 mM MOPAN was completely converted into MOPAA（9.65 mM）in 4 h in shaking flasks,with a maximal specific activity of 20.81 U/g,which was 17.16%higher than before optimization（17.24 U/g）.Moreover,kinetic studies of the nitrilase catalyzed reaction were demonstrated and the kinetic constants were as follows:K_m=0.714 mmol/l,V_m=0.061 mmol/（1·min）,K_s=13.601 mmol/l.The nitrile-degrading pathways of B.subtilis ZJB-063 were investigated and it seemed that the strain harboured versatile nitrile-amide converting activities and the two pathways of nitriles could both occur in the strain.It exhibited nitrilase activity without addition of inducers, indicating that the nitrilase in B.subtilis ZJB-063 is constitutive. Interestingly,B.subtilis ZJB-063 exhibited nitrile hydratase and amidase activity with the addition ofε-caprolactam.Moreover,the substrate spectrum altered with the alteration of enzyme systems due to the addition ofε-caprolactam.The constitutive nitrilase was high specific for arylacetonitriles,while nitrile hydratase/amidase showed a broad substrate spectrum,which catalyzed all the tested compounds.Finally,aiming at improving the reusability of the biocatalyst, alginate was employed to immobilize B.subtilis ZIB-063.After optimization in terms of mechanical strength and enzyme activity,the preparation conditions were set as follows:2.5%of sodium alginate,4% of CaCl₂,4 h of immobilization time,2 mm of bead diameter.Reaction conditions of immobilized cell-mediated conversion were evaluated.The results showed that highest conversion occurred in phosphate buffer（pH 7.0）at 32℃.The thermostability enhanced compared with the freely suspended cells,but enzyme activity decreased a bit.An initial MOPAN concentration of 10 mM was completely converted to MOPAA by entrapped cells after 7 h reaction.Efficient reusability of the biocatalyst up to 8 batches was achieved by immobilization.After the 8^thcycle,the immobilized cells retained 85%of its original activity.However,the occurrence of swelling of the beads had taken place and a disruption of a small portion of the beads was observed. Stabilization of the alginate immobilized cells with polyethyleneimine（PEI）and glutaraldehyde（GA）could alleviate the leakage of the cells.It was found that the optimum PEI,CaCl₂ and GA concentrations were 0.2%,40 mM and 0.8%,respectively,and the optimum cross-linkage time of GA was 30s.Both the tolerance to high phosphate concentration and the mechanical strength of the PEI and GA stabilized cells were considerably enhanced.Nevertheless,the stabilized beads showed a little lower activity.The data obtained revealed that 10 mM MOPAN was completely degraded by stabilized beads within 10 h. Moreover,reusability was significantly improved with retention of enzyme activity（about 68.32%）even after 17 consecutive batches. Environmental scan electron microscopy（ESEM）implied that cells after PEI treatment were more densely distributed in the matrix,which might have been ascribed to contracting of the bead by volume and thus cell density per volume increased.